Multiple structures for the 2-aminopurine-cytosine mispair

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• 2-aminopurine (2AP) is a mutagenic base analog that can induce transition mutations.
• The 2AP-cytosine (C) mispair is a key intermediate in 2AP-induced mutagenesis.
• Previous proposed structures for the 2AP-C mispair include rare tautomers, protonated forms, and wobble structures.

Purpose of the Study:

• To elucidate the structural equilibrium of the 2-aminopurine and cytosine (2AP-C) mispair.
• To understand the role of protonation and tautomerization in 2AP-C mispair formation.
• To investigate the contribution of different 2AP-C structures to 2AP-induced transition mutations.

Main Methods:

• UV spectroscopy to monitor base pair formation and protonation.
• Fluorescence spectroscopy to detect structural changes and quenching effects.
• Nuclear Magnetic Resonance (NMR) spectroscopy to analyze proton chemical shifts and exchange rates.

Main Results:

• An equilibrium was demonstrated between a neutral wobble structure and a protonated Watson-Crick structure for the 2AP-C mispair.
• The transition between these structures occurs around a pK value of 5.9-6.0.
• UV, fluorescence, and NMR data indicate predominant protonation of the 2AP residue.
• Rapid proton transfer between 2AP and C was observed in the protonated Watson-Crick configuration.

Conclusions:

• The 2AP-C mispair exists in an equilibrium between neutral wobble and protonated Watson-Crick forms.
• Protonation of 2AP is a critical step in forming the Watson-Crick-like mispair.
• Understanding this structural equilibrium is crucial for comprehending 2AP-induced transition mutations.